Preparation of samples for analysis can consume a significant quantity of the sample, cause extraneous or spurious results in the analysis, and consume significant quantities of time, thus increasing the cost involved in the analysis. Sample preparation procedures generally involve removal of salts or other undesired components present in the sample, removal of undesired solvents or exchange of one solvent in which the intended analytes are dissolved for another solvent, concentration of analytes to a predetermined concentration, and the like. Thus, inadequate sample preparation procedures can result in loss of intended analytes as well as loss of time and increased costs, rendering analytical procedures costly, time consuming, unreliable, irreproducible and unsatisfactory. Numerous methods of preparing samples are available at present, including solid phase extraction (“SPE”) to concentrate analytes from a liquid phase onto a solid phase, from which they can then be removed in relatively purer form for further analysis. Liquid phase extraction methods are also known, along with liquid-liquid phase extraction and liquid-liquid-liquid phase extraction methods.
Depending on the type of analysis to be performed, and detection method used, SPE can be tailored to remove specific interferences. Analysis of biological samples such as plasma and urine using high performance liquid chromatography (HPLC) or mass spectrometry generally requires SPE prior to analysis both to remove insoluble matter and soluble interferences, and also to pre-concentrate target compounds for enhanced detection sensitivity. Many biological samples contain salts or other ion suppressing components, which can be particularly troublesome when mass spectrometer based detection is used. SPE can also be used to perform a simple fractionation of a sample based on differences in hydrophobicity or functional groups of sample components, thereby reducing the complexity of the sample to be analyzed.
Devices designed for SPE typically include a chromatographic sorbent which allows the user to preferentially retain sample components. Once a sample is loaded onto the sorbent, a series of washing and elution fluids are passed through the device to separate contaminants or interfering compounds from intended sample analytes, and then to collect the target sample analytes for further analysis. SPE devices usually include a sample holding reservoir, a means for containing the sorbent, and a fluid conduit, or spout for directing the fluids exiting the device into suitable collection containers. The SPE device may be in a single well format, which is convenient and cost effective for preparing a small number of samples, or a multi-well format, which is well suited for preparing large numbers of samples in parallel. Multi-well formats are commonly used with robotic fluid dispensing systems. Typical multi-well formats include 48-, 96-, and 384-well standard plate formats. Fluids are usually forced through the SPE device and into the collection containers, either by drawing a vacuum across the device with a specially designed vacuum manifold, or by using centrifugal or gravitational force. Centrifugal force is generated by placing the SPE device, together with a suitable collection tray, into a centrifuge specifically designed for the intended purpose. However, all of these formats require relatively large amounts of sample and solvents, and require multiple fluid transfer steps.
Traditional SPE device designs have utilized packed beds of sorbent particles contained between porous filter discs that are contained within the SPE device. For example, U.S. Pat. No. 6,723,236 to Fisk describes SPE devices wherein sorbent particles are contained between two porous filter elements. The retention of compounds by the resulting packed beds is typically quite good, especially if the sorbent properties are carefully chosen. However, one drawback with conventional packed bed devices is that the void volume contained within the porous filters and packed bed requires that relatively large elution volumes be used to completely elute the target compounds. Typical elution volumes required to fully elute target compounds from a packed bed type SPE device fall in the range of 0.20-5 mL or more, depending on the size of the sorbent bed.
Thus, such devices are not suitable for small sample amounts or small volumes, and there is a need in the art for devices and methods for handling small sample sizes and quantities. To address these needs in the art, U.S. Pat. No. 5,906,796 to Blevins describes a solid phase extraction plate utilizing a plurality of solid phase extraction disks press fitted between the sidewalls of the chambers. A variety of extraction media were reported to be useful, in particular a nonpolar extraction medium containing silica bonded with hydrophobic groups available from Varian, Inc of Lake Forest, Calif., under the tradename SPEC®.
However, it would be convenient to incorporate solid phase extraction capabilities into microvolume liquid handling and dispensing devices themselves, thus eliminating steps in sample processing. Toward that end, U.S. Pat. No. 6,416,716 to Shukla describes a device for small sample preparation using tubes and columns such as capillaries or pipette tips in which particles of a separation medium are directly embedded in the solid material composing the device. Shukla further reports that the use of filters to hold separation media is problematic because filters slow the rate at which sample flows through the column and result in loss of sample on the filter material. Shukla states that loss of sample can be especially significant when very small sample volumes are involved. Shukla further states that filter-free columns that rely on a solid support matrix with embedded separation medium do exist, but that sample flow is low through these columns.
U.S. Pat. Nos. 6,048,457 and 6,200,474 to Kopaciewicz describe methods for preparing cast-in-place composite and/or nonfilled structures useful as sorptive or reactive media or for size based separations. The devices reportedly include a large amount of adsorptive particles entrapped in polymer while still maintaining the membrane three dimensional structure. In a preferred aspect, the methods are reported to be useful for preparing particles entrapped within a porous polymeric substrate in a pipette tip.
However, these devices and others suffer from limitations in methods of preparation that result in irreproducibility, poor flow rates, low capacity for adsorbing analytes, non-uniform flow rates, high manufacturing costs, and the like. Accordingly, there is a need in the art for improved SPE devices and methods for preparing them that overcome the limitations of the prior art devices.